Methods for Making and Delivering Rho-Antagonist Tissue Adhesive Formulations to the Injured Mammalian Central and Peripheral Nervous Systems and uses Thereof

ABSTRACT

The present invention provides methods for making, delivering and using formulations that combine a therapeutically active agent(s) (such as for example a Rho antagonist(s)) and a flowable carrier component capable of forming a therapeutically acceptable matrix in vivo (such as for example tissue adhesives), to injured nerves to promote repair and regeneration and regrowth of injured (mammalian) neuronal cells, e.g. for facilitating axon growth at a desired lesion site. Preferred active agents are known Rho antagonists such as for example C3, chimeric C3 proteins, etc. or substances selected from among known trans-4-amino(alkyl)-1-pyridylcarbamoylcyclohexane compounds or Rho kinase inhibitors. The system for example may deliver an antagonist(s) in a tissue adhesive such as, for example, a fibrin glue or a collagen gel to create a delivery matrix in situ. A kit and methods of stimulating neuronal regeneration are also included.

The present invention provides methods for making, delivering and usingformulations that combine a therapeutically active agent(s) (such as forexample a Rho antagonist(s)) and a flowable carrier component capable offorming a therapeutically acceptable matrix in vivo (such as for exa pletissue adhesives), to injured nerves to promote repair and regenerationand regrowth of injured mammalian neuronal cells, e.g. for facilitatingaxon growth at a desired lesion site, active agents are known Rhoantagonists such as for example C3, chimeric C3 proteins, etc. (seeblow) or substances selected from among knowntrans-4-amino(alkyl)-1-pyridylcarbamoylcyclohexane compounds (also seebelow) or Rho kinase inhibitors. The system for example may deliver anantagonist(s) in a tissue adhesive such as for example, a fibrin, glueor a collagen gel to create a delivery matrix in situ. A kit and methodsof stimulating neuronal regeneration are also included.

FIELD OF THE INVENTION

The present invention pertains to the field of mammalian nervous systemrepair (e.g. repair of a central nervous system (CNS) lesion site or aperipheral nervous system (PNS) lesion site), axon regeneration and axonsprouting. The present invention in particular relates to a method ofdelivery of C3 or other Rho antagonists to repair damage in the nervoussystem. The invention also pertains to use of the delivery system fortoxicity testing of compounds applied to the injured CNS. (i.e. to acentral nervous system (CNS) lesion site or a peripheral nervous system(PNS) lesion site).

In the following by way of example only reference will generally be madeto axon growth at a a central nervous system (CNS) lesion site.

BACKGROUND

Traumatic injury of the spinal cord results in permanent functionalimpairment. Most of the deficits associated with spinal cord injuryresult from the loss of axons that are damaged in the central nervoussystem (CNS). Similarly, other diseases of the CNS are associated withaxonal loss and retraction, such as stroke, HTV dementia, priondiseases, Parkinson's disease, Alzheimer's disease, multiple sclerosisand glaucoma. Common to all of these diseases is the loss of axonalconnections with their targets, and the ability to stimulate growth ofaxons from the affected or diseased neuronal population would improverecovery of lost neurological functions. For example, following a whitematter stroke, axons are damaged and lost, even though the neuronal cellbodies are alive. Treatments that are effective in eliciting sproutingfrom injured axons are equally effective in treating some types ofstroke (Boston life sciences, Sep. 6, 2000 Press release). Similarly,although the the following discussion will generally relate to deliveryof Rho antagonists, etc. to a traumatically damaged nervous system, thisinvention also pertains to damage from unknown causes, such as duringmultiple sclerosis, HIV dementia, Parkinson's disease, Alzheimer'sdisease, prion diseases or other diseases of the CNS were axons aredamaged in the CNS environment.

It has been proposed to use various agents to stimulate regeneration ofcut axons, i.e. nerve lesions. Please see for example Canadian Patentapplication nos. 2,304,981 (McKerracher et al) and 2,300,878(Strittmatter). These document propose the use of known Rho antagonistssuch as for example C3, chimeric C3 proteins, etc. (see blow) as well assubstances-selected from among knowntrans-4-amino(alkyl)-1-pyridylcarbamoylcyclohexane compounds (also seebelow) or Rho kinase inhibitors for use in the regeneration of axons.

Several major advances in our understanding of axon regeneration haveled to the ability to stimulate some axon regeneration and functionalrepair in animal models of spinal cord injury. In the 1980's experimentsby Aguayo and colleagues to use peripheral nerve grafts that wereinserted into the brain or spinal cord showed that CNS neurons have thecapacity to regrow, and these studies highlighted that diverse classesof CNS neurons have the potential to regenerate when given a permissivegrowth environment (Aguayo, et al. (1981) J Exp Biol. 95:231-40).However, this technique cannot be used to rewire the complex circuitryof the CNS. Another major advance in our understanding of axonregeneration in the central nervous system was the discovery by Schwaband colleagues that the CNS environment did not simply lack growthpromoting molecules, but that growth inhibitory molecules existed toblock axon growth (Schwab, et al. (1993) Annu. Rev. Neurosci.16:565-595). Long distance regeneration in the CNS by blocking growthinhibitory molecules with antibodies was first achieved in juvenile ratsby neutralization of inhibitory protein activity with the IN-1 antibodyin spinal cord (Schnell and Schwab (1990) Nature. 343:269-272) and opticnerve (Weibel, et al. (1994) Brain Res. 642:259-266). However, thistechnique suffers from the problem that only a single growth inhibitoryprotein is targeted, and delivery by the application of hybridoma ceilsor by infusing antibodies with pumps. There have been investigations onthe use of growth factors to promote regeneration in the CNS, some withnotable success (Ramer, et al. (2000) Nature. 403:312-316, Liu, et al.(1999) J Neurosci. 19:4370-87, Blesch, et al. (1999) J Neurosci.19:3556-66). Typically infusion pumps or gene therapy techniques areused to deliver growth factors to injured neurons. In general, trophicfactors do not stimulate long distance regeneration, but stimulate moreof a local sprouting response (Schnell, et al. (1994) Nature.367:170-173, Mansour-Robaey, et al. (1994) Proc. Natl. Acad. Sci.91:1632-1636).

A more recent advance is the demonstration that increasing the intrinsicgrowth capacity of neurons is sufficient to allow axon regeneration inthe CNS, and that neurons primed for regeneration with neurotrophins, aconditioning lesion, or treatment with Rho antagonists have a betterchance to grow on inhibitory substrates (Neumann (1999) Neuron.23:83-91, Cai, et al. (1999) Neuron. 22:89-101, Lehmann, et al. (1999)J. Neurosci. 19:7537-7547). Targeting intracellular signallingmechanisms is likely to be the most efficient way to promote axonregeneration, and it has been found that Rho antagonists are able tostimulate regeneration in the optic nerve of adult rats (Lehmann et al(1999) IBID). However, preliminary experiments to apply Rho antagoniststo the injured spinal cord were not successful. It is believed that theinfused protein was not sufficiently retained at the injury site, eitherby syringe application or the use of Gelfoam. This suggested that thedelivery of compounds that act with low affinity (compared to highaffinity neurotrophins) posed unique problems in delivery. As shall bediscussed in greater detail below the present invention relates to atissue-adhesive delivery system whereby the Rho antagonist is added tothe adhesive solution before application of the solution with a syringe,and polymerization of the adhesive within the lesion cavity in the CNS.

While neurons in the peripheral nervous system regenerate naturally,there are many techniques used to enhance and help the repair process.Most of these techniques are not aimed at stimulating the rate of axonalregeneration, but in helping to guide axons back towards their targetregions. For example, severed nerve are sewn or glued together with afibrin glue enhance the repair process. While the following discussionwill generally relate or be directed at repair in the CNS, thetechniques described herein may be extented to use in PNS repair.Treatment with Rho antagonists in the adhesive delivery system could beused to enhance the rate of axon growth in the PNS. This is first use ofRho antagonists in the PNS.

Growth inhibitory proteins cause growth cone collapse (Li, et al. (1996)J. Neurosci. Res. 46:404-414, Fan, et al. (1993) J. Cell Biol.121:867-878) and it has become clear that GTPases of the Rho family thatcomprise Rho, Rac and Cdc42 are intracellular regulators of growth conecollapse (Lehmann, et al. (1999)J. Neurosci. 19:7537-7547, Tigyi, et al.(1996) Journal of Neurochemistry. 66:537-548, Kuhn, et al. (1999) J.Neurosci. 19:1965-1975, Jin and Strittmatter (1997) J. Neurosci.17:6256-6263).These small GTPases exist in inactive (GDP) and active(GTP) forms, and the cycling between active GTP-bound and inactiveGDP-bound states is tightly regulated. The guanine nucleotide exchangefactors (GEFs) accelerate the release of GDP, thereby facilitating GTPbinding. The GTPase activating proteins (GAPs) catalyze GTP hydrolysisand conversion of the inactive form. The GDP dissociation inhibitors(GDIs) act to maintain Rho in a GDP-bound form. GEFs for Rho all have adomain homologous with the Dbl oncoprotein, and more than 20 suchproteins have been identified, including Tiam-1 which is highlyexpressed in brain (Zheng and Li (1999) J. Biol. Chem. 272:4671-4679,van Leeuwen, et al. (1997) J. Cell Biol. 139:797-807). Once in theactive form, Rho GTPases typically stimulate ser/thr kinases, such asROK (Rho kinase), PAK (p21-activated kinase) and downstream effectorsthat act on the cytoskeleton.

The Rho family members that regulate the cytoskeleton and motilityinclude Rho, Rac and Cdc42 (Nobes and Hall (1995)Cell 1995.81:53-62).Rho is an important link between signaling through integrins andsignaling cascades of trophic factors (Laudanna, et al. (1996) Science.271:981-983, Hannigan, et al. (1996) Nature. 379:91-96, Kuhn, et al.(1998) J. Neurobiol. 37:524-540). Cdc42 is important for the regulationof filopodia (Nobes and Hall (1995) Cell 1995.81:53-62). Both Rac andRho regulate growth cone motility and axon growth. In non-neuronal cellsa hierarchy of signaling between Rho, Rac and Cdc42 exists (Hall (1996)Ann. Rev. Cell Biol. 10:31-54). In neurons Rac and Rho may have oppositeeffects (van Leeuwen, et al. (1997)J. Cell Biol_139:797-807, Kozma, etal. (1997) Molec. Cell. Biol. 17:1201-1211). Activation of Racstimulates outgrowth of neurites from N1E-115 neuroblastoma neuronswhereas activation of Rho causes neurite retraction (van Leeuwen, et al.(1997) J. Cell Biol. 139:797-807, Albertinazzi, et al. (1998)J. CellBiol. 142:815-825). In PC12 cells, dominant negative Rac disruptsneurite outgrowth in response to NGF (Hutchens, et al. (1997)Molec.Biol.Cell. 8:481-500, Daniels, et al. (1998) EMBO Journal.17:754-764) . whereas treatment of PC12 cells with lysophosphatidic acid(LPA), a mitogenic phospholipid that activates Rho, causes neurite.retraction (Tigyi, et al. (1996) Journal of Neurochemistry. 66:537-548).The p21-activated kinase(PAK) is activated by Rac, and PAK can alsoinduce PC12 cell neurite outgrowth (Daniels, et al. (1998) EMBO Journal.17:754-764). It has been shown that inactivation of Rho is sufficient topromote PC12cell neurite outgrowth on growth inhibitory substrates(Lehmann, et al. (1999) J. Neurosci. 19:7537-7547). A recent study ofactivating and null mutations of Rho expressed in PC12 cells suggeststhat the differentiation state is an important parameter for the effectof Rho on neurite outgrowth, and that priming PC12 cells with NGF canalter the responsiveness to activating and null mutations (Sebok, et al.(1999) J. Neurochem. 73:949-960). This result is in agreement with thefinding that priming neurons increases intracellular cAMP (Cai, et al.(1999) Neuron. 22:89-101), which can in turn influence the activation ofRho (Lang, et al. (1996) EMBO J. 15:510-519, Dong, et al. (1998) J.Biol. Chem. 273:22554-22562).

In primary neurons Rac and Rho regulate both dendrite and axon growthand cone morphology and collapse. By immunocytochemistry it has beendemonstrated that Rho is concentrated in growth cones, and somecolocalizes at sites of point contact (Renaudin, et al. (1998) J.Neurosci. Res. 55:458-471). Experiments with activating and dominantnegative mutations have demonstrated that activation of Rac is importantin maintaining a spread morphology after challenge with growth conecollapsing factors (Kuhn, et al. (1999) J. Neurosci. 19:1965-1975, Jinand Strittmatter (1997) J. Neurosci. 17:6256-6263). The activation ofRho induces growth cone collapse, and collapse can be prevented bytreatment with Clostridium botulinum C3 exotransferase (hereinaftersimply referred to as C3) (Tigyi, et al. (1996) Journal ofNeurochemistry. 66:537-548, Jin and Strittmatter (1997) J. Neurosci.17:6256-6263). C3 inactivates Rho by ADP-ribosylation and is fairlynon-toxic to cells (Dillon and Feig (1995)Methods in Enzymology: SmallGTPases and their regulators Part. B. 256:174-184).

An important downstream target of activated Rho is p160ROK, a Rho kinase(Kimura and Schubert (1992) Journal of Cell Biology. 116:777-783,Keino-Masu, et al. (1996) Cell. 87:175-185, Matsui, et al. (1996) EMBO115:2208-2216, Matsui, et al. (1998) J. Cell Biol. 140:647-657, Ishizaki(1997) FEBS Lett. 404:118-124). Among other effects, ROK phosphorylatesmyosin phosphatase to regulate actin-myosin based motility (Matsui, etal. (1996) EMBO J. 15:2208-2216) and regulates proteins of the ezrinfamily (Vaheri, et al. (1997) Curr. Opin. Cell Biol. 9:659-666), whichare concentrated in neuronal growth cones (Goslin, et al. (1989) J. CellBiol. 109:1621-1631). Activation of ROK also induces growth conecollapse, which can be prevented by the addition of the ROK inhibitorY-27632 (Hirose, et al. (1998) J. Cell Biol. 141:1625-1636).

The above studies showed that Rho antagonists can stimulate regenerationin the CNS. It has been demonstrated that Rho kinase is an importantdownstream target of Rho signaling (Matsui, et al. (1996) EMBO J.15:2208-2216, Bito (2000) Neuron. 26:431-441). Among other effects,inactivation of Rho kinase stimulates neurite outgrowth in tissueculture (Bito (2000) Neuron. 26:431-441) as does inactivation of Rho(Lehmann, et al. (1999) J. Neurosci. 19:753 7-7547). Therefore,inactivation of Rho kinase should induce the same biological effects invivo as inactivation of Rho.

The Rho kinase inhibitory Y-27632 compound is atrans-4-amino(alkyl)-1-pyridylcarbamoylcyclohexane compound; thiscompound is for example described in U.S. Pat. No. 4,997,834 the entirecontents of which are incorporated herein by reference; this patentrefers for example to compounds which may be selected from the groupconsisting of trans-4-aminomethyl-1-(4- pyridylcarbomoyl)cyclohexane,trans-4-aminomethyl-trans-1 -methyl-1-(4-pyridylcarbamoyl)cyclohexane,trans-4-aminomethyl-cis-2-methyl-1-(4-pyridylcarbamoyl)cyclohexane,trans-4-aminomethyl-1-(2-pyridylcarbamoyl)cyclohexane,trans-4-aminomethyl-1-(3-pyridylcarbamoyl)cyclohexane,trans-4-aminomethyl-1-(3-hydroxy-2-pyridylcarbamoyl)cyclohexane,trans-4-aminomethyl-1-(3-methyl-4pyridylcarbamoyl)cyclohexane,4-(trans-4-aminomethylcyclohexylcarboxamido)-2,6-dimethyl-pyridine-N-oxide,trans-4-aminomethyl-1-(2-methyl-4-pyridylcarbamoyl)cyclohexane,trans-4-(2-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane,trans-4-(1-amino-1-methylethyl)-1-(4-pyridylcarbamoyl)cyclohexane,trans-4-(1-aminopropyl)-1-(4-pyridylcarbamoyl)cyclohexane, andpharmaceutically acceptable acid addition salts thereof.

Please also see also Ishizali et al. 2000. Molecular Pharmacology57:976-983 3 which refers to Y-27632 in the dihydrochloride form as wellas to a related compound Y-30141, namely(R)-trans-4-(1-aminoethyl)-N-(1H-pyrrolo[2,3]pyridin-4-yl)cyclohexanecarboamide dihydrochloride. A patent application A Medicinescomparising Rho kinase inhibitor has been submitted (EPO 956 865 A1).This inhibitor has not been tested for efficacy in CNS injury, nor hasthe company who patented this compound discovered how it might toapplied to a region of CNS injury in a kit form. Such a kit is providedin our invention. Please see also European Patent application no.97934756.4; PCT/JP97/02793; International publication # WO 98/06433(19.02.1998/07).

The compound Y-27632 has the following structure

The above structrure is used herein in a pharmaceutically aceptable saltform (e.g dihydrochloride salt).

The above mentioned related compound Y-30141 which may be exploited inaccordance with the present invention has the following structure:

Again the above structrure may also be used herein in a pharmaceuticallyaceptable salt form (e.g. dihydrochloride salt).

The compound(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboamide(Y-27632) inhibits Rho kinase at sub-micromolar concentrations (Uehata,et al. (1997) Nature. 389:990-994). Y-27632, made by a Yoshitoma,affects calcium sensitization of smooth muscles to affect hypertension.It was reported that the cellular target of Y-27632 is Rho-associatedprotein kinase, p160ROCK (Uehata, et al.;(1997) Nature. 389:990-994,Somlyo (1997) Nature. 389:908-911).

Different methods have been used for local delivery of drugs in the CNS,however none of these methods have been developed as a kit withbiological component that have proven effective in the promotion of theregeneration of injured axons. IN-1 is an antibody that , promotesregeneration in the CNS. One method of delivery is the implantation ofcells that secrete the active antibody (Schnell et al (1994) Nature367:170). The use of fibrin adhesive for the delivery of IN-1 antibodywas not found to be effective (Guest (1997) J. Neurosci. Res.50:888-905). Another method is the use of pumps to infuse and delivercontinuously over time compounds that stimulate regeneration. (Ramer, etal. 2000, Nature. 403:312-316, Verge, et al. 1995. Journal ofNeuroscience. 15:2081-2096).

Fibrin adhesives per se have been used in studies of CNS regeneration.It has been used in replacement of sutures to graft peripheral nervesinto the damaged CNS (Cheng, et al. (1996) Science. 273:510-513). Afibrin glue has also been used for the delivery of fibroplast growthfactor (FGF) to damaged corticospinal neurons (Guest (1997) J. Neurosci.Res. 50:888-905). The use of fibrin glue plus FGF did not promote longdistance regeneration.

Collagen per se has been tested for its ability to promote regenerationafter injury (Joosten (1995) J. Neurosci. Res. 41:481-490.). Collagenhas also been used for the delivery of neurotrophins to injuredcorticospinal axons (Houweling (1998) Expt. Neurol. 153:49-59). Neitherof the conditions was able to support long distance regeneration. Intissue culture, collagen gels can maintain gradients of small moleculesimportant in axon guidance (Kennedy, et al. (1994) Cell. 78:425-435).Moreover, it had been reported that collagen gels by themselves couldfoster some axon regeneration after spinal-cord injury (Joosten (1995)J. Neurosci. Res. 41:481-490).

Many different protein-based tissue adhesives exist Examples includecollagen gels, fibrin tissue adhesives, matrigel, laminin networks, andadhesives based on a composition of basment membrane proteins thatcontain collagen; Perhaps the most popular are the fibrin adhesives.

Fibrin sealant has three basic components: fibrinogen concentrate,calcium chloride and thrombin. Other components can be added to affectthe properties of the gel formation. Added components are used tomodulate time it takes for the fibrin gel to form from the solublecomponents, the size of the protein network that is formed, the strengthof the gel, and protease inhibitors slow down the removal of the gelafter it is place in the body. Several different commercial preparationsare available as kits. These include Tissucol/Tisseel, (Immuno AG,Vienna, now marketed by Baxter), Beriplast P, (Hoechst, West Germany),and Hemaseel (Hemacure Inc. Kirkland, Quebec).

To make a fibrin gel soluble thrombin and fibrinogen are mixed in thepresence of calcium chloride. When the components mix, a fibrin adhesivegels is formed because the fibrinogen molecule is cleaved by thrombin toform fibrin monomers. The fibrin monomers spontaneously will polymerizeto form a three-dimensional network of fibrin, a reaction that mimicsthe final common pathway of the clotting cascade, i.e. the conversion offibrinogen to fibrin sealant. The key to the preparation of commercialpreparations is to keep the frinogen and thrombin components separateuntil use, so that the poymerization can be controlled with the desiredtiming before or after application to the body.

Today such use of fibrin as a biologic adhesive has been widely acceptedand found application in many fields of surgery. HEMASEELJ or Tisseel VHare used as an adjunct to hemostasis in surgeries involvingcardiopulmonary bypass and treatment of splenic injuries due to blunt orpenetrating trauma to the abdomen, when control of bleeding byconventional surgical techniques, including suture, ligature and cauteryis ineffective or impractical. The action iof these fibrin gels is alsoused to stop bleeding in surgical procedures involving cardipulmonarybypass and repair of the spleen. Tisseel VH has also been shown to be aneffective sealant as an adjunct in the closure of colostomies.

Collagen gels have been used in tissue culture studies to main gradientsof diffusible molecules. The use of collagen gels has permitted theidentification and testing of neuronal guidance factors such as netrins(Kennedy, et al. (1994) Cell. 78:425-435). When collagen polymerized itforms a dense protein network. Therefore, like fibrin, it has thepotential to act as a tissue adhesive. Moreover, collagen is easy topurify in large quantities.

There are many different types of collagens, and it is a major componentof basement membranes in many different body tissues. The form ofcollagen often used for experimental studies in rodents is type IVcollagen because it is easily purified from rat tails.

Not only is collagen a component of the basement membrane in theperipheral nervous system, but it is known that neurons expressreceptors for collagen. Receptors for collagens are receptors of theintegrin class of proteins. One important collagen receptor expressed byneurons is the alph1 beta1 receptor (McKerracher, et al. 1996. Molec.Neurobiol. 12:95-116); this receptor is involved in the promotion ofneurite outgrowth. When PC12 cells, a neuronal cell line, are plated oncollagen substrates in tissue culture, collagen helps promote neuritegrowth in an integrin-dependent fashion. The addition of anit-integrinantibodies block neurite ourgrowth. Therefore, the ability of collagen,by itself, has been tested for its ability to promote axon regenerationafter spinal cord injury. It was reported that collagen gels bythemselves could foster some axon regeneration after spinal cord injury(Joosten (1995) J. Neurosci. Res. 41:481-490.). However, the observedgrowth was more of a sprouting response with out any long distanceregeneration past the glial scar and site of the lesion. In addition,collagen has been tested for its ability to promote regeneration afterinjury in conjunction with the delivery of neurotrophins to injuredcorticospinal axons (Houweling (1998) Expt. Neurol. 153:49-59). Thistreatment was not able to support long distance regeneration, althoughtthe treated animals had a better sprouting response than the controls.

It would be advantageous to have a means for the direct delivery to andmaintenance at a lesion site of an agent able to facilitate axon growthat the lesion site.

SUMMARY OF THE INVENTION

As discussed herein in acccordance with the present invention atherapeutically active agent for facilitating axon growth may bedelivered (in a flowable matrix forming substance) to a (nerve) lesionsite, for example, by injection using known syringe type glue or sealantdevices modified as necessary or desired (e.g. by addition of a furthersubstance container); examples of known delivery devices, systems,mechanisms, matrix forming compositions, and teh like are shown forexample in U.S. Pat. No. 5,989,215, U.S. Pat. No. 4,978,336, U.S. Pat.No. 4,631,055, U.S. Pat. No. 4,359,049, U.S. Pat. No. 4,974,368, U.S.Pat. No. 6,121,422, U.S. Pat. No. 6,047,861, U.S. Pat. No. 6,036,955,U.S. Pat. No. 5,945,1115, U.S. Pat. No. 5,900,408, U.S. Pat.No.6,124,273, U.S. Pat. No. 5,922,356, and in particular U.S. Pat. No.6,117,425; the entire contents of each of these patents is incorporatedherein by reference.

A sufficient amount of a therapeutically active agent for facilitatingaxon growth may be dispersed in a stable flowable (known) type of(proteinaceous) matrix forming material. Once delivered to the desiredlesion site the resulting in situ or in vivo matrix (e.g. gel orcrosslinked substances) inhibits the migration or diffusion of the agentfrom the site of injection, so as to maintain the primary effect of theagent in the region of injection, i.e. in the area of the lesion. In anyevent the active agent is to be present in an amount effective tofacilitate axon growth.

A substantially uniform dispersion of the active agent may be initiallybe formed so as to provide a concentrated amount of active agent in aphysiologically acceptable matrix forming material. The matrix formingmaterial may be comprised of any (known) individual or combination ofpeptides, proteins etc. which provides for stable placement, orcombinations thereof. Of particular interest is a collagen material, afibrinogen material, or derivatives thereof; other high molecular weightphysiologically acceptable biodegradable protein matrix formingmaterials may if desired be used. The active agent may, for example, beincorporated in a sufficient concentration so as to provide the. desiredor effect the desired sustained release.

Typically when estimating doses in different animal species, the sameweight ratio is used. It is for example possible to apply 40 ug proteinper 20 gm mouse. Therefore, we anticipate that the ideal dose should beapproximately 3 gm per 60 kg person. We expect that the dose necessarywill depend on the size of the lesion and the time of application (acuteor chronic) spinal cord injury. In cases of chronic injury, there isoften a necrotic center in the spinal cord, and higher doses may berequired.

The matrix forming material may be a one-component adhesive or sealanttype material (e.g. collagen material); alternatively it may be amult-component adhesive or sealant (e.g. a fibrinogen based material).The matrix may be a human protein matrix or if necessary or desired anon-human protein matix; preferably a human protein matrix.

The (proteinaceous) matrix forming material is flowable for injection,but once in vivo it provides for stable placement, of the active agentin the lesion area; i.e. after injection, the active agent is releasedinto the immediate environment the matrix providing a medium forprolonged contact between a lesion site and the active agent (i.e. axongrowth facilatator or stimulant).

The matrix forming material(s) is (are) of course to be chosen on thebasis that the materials and resultant formed matrix will be capable onthe one hand of holding the active agent for release in situ and on theother without preventing the therapeutic effect thereof, i.e. the matrixis to be therapeutically acceptable. The choice of active agent may bedetermined empirically through appropriate or suitable assays keeping inmind that the matrix etc. are to to be therapeutically acceptable.

The present invention in an aspect relates to a biocompatible,(supplemented tissue sealant or adhesive) composition comprising: (i) atleast one supplement selected from the group consisting oftherapeutically active agents for facilitating axon growth; and (ii) aflowable carrier component capable of forming a pharmaceutically ortherapeuticallly acceptable matrix (in vivo)—i.e. a nerve lesion site;wherein said supplement is releasable from said matrix into the adjacentexternal environment (e.g. for a sustained period of time).

The present invention in another aspect relates a method for thepreparation of a flowable biocompatible composition comprising admixing(i) at least one supplement selected from the group consisting oftherapeutically active agents for facilitating axon growth and (ii) aflowable carrier component capable of forming a therapeuticalllyacceptable matrix in vivo at a nerve lesion site; wherein saidsupplement is releasable from said matrix into the adjacent externalenvironment.

By way of example only in accordance with the present invention a methodof applying an supplemented solution of polymerizable fibrin to adesired lesion site, may comprise a) affixing a cartridge containingimmobilized thrombin to a syringe containing a solution of r fibrinogen,b) contacting the solution of fibrinogen with immobilized thrombin underconditions resulting in an activated solution of polymerizable fibrin bypassing the solution of fibrinogen through the cartridge containingimmobilized thrombin, c) adding to the fibrinogen solution or to theactivated solution a supplement (i) at least one supplement selectedfrom the group consisting of therapeutically active agents forfacilitating axon growth; and c) delivering the supplemented activatedsolution of polymerizable fibrin to the desired lesion site (e.g. acentral nervous system (CNS) lesion site or a peripheral nervous system(PNS) lesion site) under conditions which result in polymerized fibrinat the lesion site having dispersed therein the supplement wherein saidsupplement is released from said fibrin matrix into the adjacentexternal environment.

In accordance with another aspect the present invention relates to a kitcomprising, in suitable container means (e.g. separate means): (a) afirst pharmaceutical composition or substance comprising a biologicalagent capable of facilitating axon growth; and (b) a secondpharmaceutically or therapeutically acceptable component comprising asingle flowable carrier component or two or more separate componentscapable once intermingled of forming a flowable carrier component, saidflowable carrier components each being capable of forming apharmaceutically or therapeutically acceptable matrix (e.g.proteinaceous matrix, i.e. a proteinaceous glue, proteinaceous sealant,proteinaceous gel, etc.; e.g. a human derived proteinaceous matrix) invivo at a (nerve) lesion site.

In particular the present invention provides a (axon growth stimulation)kit comprising a) a first container means (e.g. one or more separatecontainers) for containing a flowable carrier component(s) or two ormore separate components capable once intermingled of forming a flowablecarrier component, said flowable carrier components each being capableof forming a pharmaceutically or therapeutically acceptable matrix (e.g.proteinaceous matrix, i.e. a proteinaceous glue, proteinaceous sealant,proteinaceous gel, etc.; ie.g. a human derived proteinaceous matrix) invivo at a (nerve) lesion site (e.g. a central nervous system (CNS)lesion site or a peripheral nervous system (PNS) lesion site) and

b) a second container means for containing a therapeutically activeagent for facilitating axon growth at the lesion site

wherein said therapeutically active agent supplement is releasable fromsaid in vivo matrix into the adjacent external environment (e.g. for asustained period of time). Alternatively, if desired or as necessary,the first and second container means may be the same, (e.g. a containermay hold collagen and C3). The kit may if desired or necessaryadditionally comprise means for dispersing (i.e. co-mingle, blend, etc.)the therapeutically active agent in said flowable carrier component soas to form a flowable axon growth stimulation . composition as well asmeans for delivering the flowable axon growth stimulation composition tothe lesion site (e.g. syringe needle). The pharmaceutically acceptablematrix may as discussed herein be a collagen matrix or a fibrin matrix.

In accordance with the present invention the therapeutically activeagent for facilitating axon growth may for example be a Rho antagonistwhich may be identified by an assay method comprising the followingsteps:

a) culturing neurons on inhibitory substrate or a substrate thatincorporates a growth-inhibitory protein.

b) Exposing the cultured neuron of step a) to a candidate Rho antagonistin an amount and for a period sufficient to permit growth of neurites,and determining if the candidate has elicited neurite growth from thecultured neurons of step a), the appearance of neurites being suggestiveor indicative of a Rho antagonist.

A compound can be confirmed as a Rho antagonist in one of the followingways:

a) Cells are cultured on a growth inhibitory substrate as above, andexposed to the candidate Rho antagonist;

b) Cells of step a) are homogenized and a pull-down assay is performed.This assay is based on the capability of GST-Rhotektin to bind toGTP-bound Rho. Recombinant GST-Rhotektin or GST rhotektin binding domain(GST-RBD) is added to the cell homogenate made from cells cultured asina). It has been found that inhibitory substrates activate Rho, andthat this activated Rho is pulled down by(GST-RBD). Rho antagonists willblock activation of Rho, and therefore, an effective Rho antagonist willblock the detection of Rho when cell are cultured as described by a)above;

c) An alternate method for this pull-down assay would be to use theGTPase activating protein, Rho-GAP as bait in the assay to pull downactivated Rho, as described (Diekmann and Hall, 1995. In Methods inEnzymology Vol. 256 part B 207-215).

Another method to confirm that a compound is a Rho antagonist is asfollows: When added to living cells antagonists that inactivate Rho byADP-ribosylation of the effector domain can be identified by detecting amolecular weight shift in Rho (Lehmann et al, 1999 Ibid). The molecularweight shift can be detected after treatment of cells with Rhoantagonist by homogenizing the cells, separating the proteins in thecellular homogenate by SDS polyacrylamide gel electrophoresis. Theproteins are transferred to nitrocellulose paper, then Rho is detectedwith Rho-specific antibodies by a Western blotting technique.

Another method to confirm that compound is a Rho-kinase antagonist is asfollows:

a) Recombinant Rho kinase tagged with myc epitope tag, or a GST tag isexpressed in Hela cells or another suitable cell type by transfection.

b) The kinase is purified from cell homogenates by immunoprecipationusing antibodies directed against the myc tag or the GST tag.

c) The recovered immunoprecipitates from b) are incubated with [32P] ATPand histone type 2 as a substrate in the presence or absence of the Rhokinase. In the absence of Rho kinase activity the Rho kinase antigens isable to block the phosphorylation activity of Rho kinase (i.e.phosphorylation of hislore), and as such identified the compound as aRho kinase antagonist.

The present invention is in particular, concerned with a delivery systemand kit to apply for example, known C3, chimeric C3, or Y-27632 typecompounds (e.g. Y-27632, Y-30141 and the like) or a Rho kinase inhibitorto injured regions of the CNS that include injured spinal cord or brain,and regions of the CNS injured by stroke. The nature of C3 is discussedherein; Y-27632 is for example mentioned above.

In the context of the present invention, the ability of C3 to stimulate(axon) regeneration in vivo was examined. Thus adult rat optic nerveswere crushed an C3 applied at the same time, directly at the lesion site(Lehmann, et al. (1999) J. Neurosci. 19:7537-7547). It was found thatlarge numbers of axons traversed the lesion to grow in the distal opticnerve. In particular there was for example examined the delivery of C3to optic nerve through the use of gelfoam an Elvax, a slow releasematrix (Lehmann, et al. (1999) J. Neurosci. 19:7537-7547).

It has also been found that the combination of collagen gels and C3 wasable to allow axons to into the site of the glial scar. Based onexperiments with fibrin glue (see below), it is believed that deliveryof C3 in collagen may be improved by the addition of protease inhibitorsto prevent lysis of the gel and C3.

However, the present invention as mentioned above is directed to thedelivery system of a therapeutically active agent (such as for example aRho antogonist—C3, Y-27632, etc.) in a protein matrix that holds theactive agent (e.g. Rho antogonist) at the site of application. Thisdelivery system retains the active agent (e.g. Rho antagonist) at thesite of CNS injury, allows large doses to be given at the site of injuryand prevents large amounts of the active agent (e.g. Rho antagonist)from leaking into the systemic circulation. The protein matrix caneither be based on the fibrin, a protein of the coagulation pathway, orit can be based on collagen, a protein of the extracellular matrix. Bothproteins when applied under specific conditions form protein networkswhen polymerized. These proteins can be applied in soluble form with theadditional components necessary for polymerization, together with theRho antagonist. When the components are mixed immediately before use,polymerization occurs after application to the body site, in our caseafter application t the CNS.

The present invention as mentioned above in particular relates to a kitsuitable for use in the above-described method of delivering fibrinsealant components to a wound site. The kit comprises individuallypackaged component solutions provided in separate bottles to Dreventmixing before use, and an applicator designed so as to permit mixing ofthe fibrinogen/Factor XIII and thrombin with C3 at the body site. Thekit provides pre-measured amounts of the fibrinogen and factor XIII inone bottle, the thrombin in another bottle, a C3 solution in anotherbottle. The contents of the bottles would be mixed in a prescribedorder, as detailed in the example below. The kit can also include one ormore other storage containers which are any necessary reagents includingsolvents, buffers, calcium chloride, protease inhibitors etc. The kitcould be sold as lyophilized or frozen components to preserve theactivity of C3or other Rho antagonist added to the kit.

Rho antagonist delivery system may be used in conjunction celltransplantation. Many different cell transplants have been extensivelytested for their potential to promote regeneration and repair. Theseinclude, but are not restricted to, Schwann cells (Xu, et al. (1996)Exp. Neurol. 134:261-272, Guest (1997) Exp. Neurol. 148:502-522.,Tuszynski, et al. (1998) Cell Transplant. 7:187-96), fibroblastsmodified to express trophic factors (Liu, et al. (1999) J Neurosci.19:4370-87, Blesch, et al. (1999) J Neurosci. 19:3556-66, Tuszynski, etal. (1994) Exp Neurol. 126:1-14, Nakahara, et al. (1996) CellTransplant. 5:191-204), fetal spinal cord transplants (Diener andBregman (1998) J. Neurosci. 18:779-793, Bregman (1993)Exp. Neurol.123:2-16), macrophages (Lazarov-Spiegler, et al. (1996)FASE B. J.110:1296-1302), embryonic stem cells (McDonald, et al. (1999) Nat Med.5:1410-2) , and olfactory ensheathing glia (Li, et al. (1997)Science.277:2000-2002, Ramon-Cueto, et al. (1998) J Neurosci.18:3803-15, Ramon-Cueto, et al. (2000) Neuron.25:425-35).

BRIEF DESCRIPTION OF THE FIGURES WHICH ILLUSTRATE EXAMPLE EMBODIMENTS OFTHE PRESENT INVENTION

FIG. 1A is a schematic diagram of adhesive delivery system of C3 appliedto an injured spinal cord wherein a tissue adhesive plus Rho antagonist(i.e. C3) is njected into the site of ninjury;

FIG. 1B is a schematic diagram of adhesive delivery system of C3 appliedto an injured spinal cord wherein the injection is shown as resulting inaxon regeneration thrugh the supplemented adhesion matrix and into thedistal spinal cord;

FIG. 2 Schematically illustrates the model used to show efficacy invivo. A dorsal hemisection was made in adult mice. Three to four weekslater the anterograde tracer WGA-HRP was injected into the cortex tolabel the neurons of the corticospinal tract. Two days later the spinalcord was removed and and HRP enzymatic activity revealed to detect theCST axons. The corticospinal tract of adult mice was lesion at the T6level, and the fibrin glue/C3 was added at the time of lesion with asyringe. The expression CST referes to cortical spinal tract.

FIG. 3 Illustrates a longitudinal section of an untreated adult mousespinal cord 3 weeks after lesion of the corticospinal tract viewed bydarkfield microscopy. The fibres were anterogradely labeled from themotor cortex and appear fluorescent. The fibres retract back from thesite of the lesion and do not regenerate with treatment.

FIG. 4A Illustrates a low magnification view of a control animal treatedwith collagen gel without C3; axons retract from the site of lesion;

FIG. 4B Illustrates a higher magnification view of a spinal cord treatedwith collagen gel without C3; axons do not regenerate;

FIG. 4C Illustrates a low magnification view of labelled corticospinalaxons near the lesion site after treatment with collagen gel with C3 asa Rho antagonist; axons do not retract bak from the lesion site; thyextend into the region of increased cellularity which is the scar;

FIG. 4D Illustrates a higher magification view of FIG. C showing thattreatment with Rho antagonst is a collagen gel allows some axons tosprout into the lesion site;

FIG. 5A Illustrates a low magnification view of a spinal cord followingtreatment with fibrin adhesive with C3 as a Rho antagonist; the sectionis viewed by darkfield to show the anterogradley-labeled fibres thatappear white;

FIG. 5B Illustrates a hgh magnification view of the lesion ate shown inFIG. 5A showing that axons grow through the scar region; the scarappears as the verticle line;

FIG. 5C Illustrates a hgh magnification view approximately 7 mm distal othe lsion ste of the spinal cord shown in FIGS. 5A and 5B; theregenerating fibres (arrows) grow long distances;

FIG. 6A Illustrates a darkfield microscopy of a spinal cord sectionafter treatment with Rho antagnist C3 in a fibrin adhesive showning longdistance regeneration; axons sprout into the white matter and cross thelesion site;

FIG. 6B Illustrates a section of the same spinal cord shown in FIG. 6Ato show axons that have regenerated a distance of 10 mm from the lesionsite;

FIG. 7A Illustrates an untreated mose two days after spinal cord injury;the control mouse is mobile but uses its front paws to drag itselfforward and it shows some movement of hindlimb joints;

FIG. 7B Illustrates an animal 2 days after spinal cord injury andtreatment with C3/matrix; the animal is able to walk with weight supporttwo days after treatment;.

FIG. 7C Illustrates a comparison of fibrin, collagen, Gelfoam and Elvaxmethods of C3delivery on long-distance regeneration. Animals weretreated with the test delivery system without (−C3) or with (+C3) Rhoantagonist. Distance of growth of the longest axon was scored by blindexamination of at least five sections from each animal. The longestdistance of axon growth was scored. Not shown here is that the animalsthat were not treated with Rho antagonist always showed axon retractionback from the site of lesion. When axon growth was measured, thedistance was measured from from the proximal edge of the lesion site.Each point represents data from one animal (approximately 5 sections peranimal);

FIG. 8 Is illustrative of open field test of behavioral recovery. Micewere scored for recovery of function by the 21 point BBB open field test(see experimental section). Two phase of recovery are. seen. An earlyphase, observed in all mice, although the BBB score is higher in the C3-treated mice. The later phase of recovery of coordinatedforelimb-hindlimb movement was only observed after treatment with C3.The C3-treated mice regain almost normal walking behavior; and

FIG. 9 Is a schematic diagram of a system exploiting a kit in accordancewith the present invention.

As used herein it is to be understood that a number of words and/orexpressions are to have the meanings as hereinafter described.

The term “fibrin glue” or “fibrin clot” is meant to include anyformulations used to make a fibrinclot: eg usseel VH or see (Herbert(1998) J. Biomed. Mater Res. 40:551-559, Cheng, et al. (1996) Science.273:510-513, Guest (1997) J. Neurosci. Res. 50:888-905). Anotherdefinition is any fibrin glue composition not sold as Tisseel, but madeby combining fibrinogen, thrombin calcium ions, with or without othercomponents such as factor XIII or apoprotinin.

The term “Rho antagonists” includes, but is not restricted to (known)C3, including C3chimeric proteins, Y276321, or other Rho antogonistsdelivered in the delivery system.

The term “Y276321” is defined as a Rho kinase inhibitor that stimulatedneurite outgrowth through its ability to inactive the Rho signalingpathway (Uehata, et al. (1997) Nature. 389:990-994, Bito(2000)Neuron.26:431-441).

The term “nerve injury site” refers to a site of traumatic nerve injuryor nerve injury caused by disease. The nerve injury site may be a singlenerve (eg sciatic nerve) or a nerve tract comprised of many nerves (eg.damaged region of the spinal cord). The nerve injury site may be in thecentral nervous system of peripheral nervous system in any regionneeding repair. The nerve injury site may form as a result of damagecaused by stroke. The nerve injury site may be in the brain as a resultof surgery, brain tumour removal or therapy following a cancerouslesion. The nerve injury site may result from Parkinson's disease,Alzheimer's disease, Amyotrophic lateral sclerosis, diabetes or anyother type of neurodegenerative disease.

Rho GTPases include members of the Rho, Rac and Cdc42 family ofproteins. Our invention concerns Rho family members of the Rho class.Rho proteins consist of different variants encoded by different genes.For example, PC12 cells express RhoA, RhoB and RhoC (Lehmann et al 1999IBID). To inactivate Rho proteins inside cells, Rho antagonists of theC3 family type are effective because they inactivate all forms of Rho(eg. RhoA Rho B etc). In contrast, gene therapy techniques, such asintroduction of a domainant negative RhoA family member into a diseasedcell, will only inactivate that specific RhoA family member.

Compounds of the C3 family from closteridium botulinum inactivate Rho byADP-ribosylation.

Recombinant C3 proteins, or C3 proteins that retain the ribosylationactivity are also effective in our delivery system and are covered bythis invention. In addition, Rho kinase is a well-known target foractive Rho, and inactivating Rho kinase has the same effect asinactiving Rho, at least in terms of neurite or axon growth (Kimura andSchubert (1992) Journal of Cell Biology. 116:777-783, Keino-Masu, et al.(1996) Cell. 87:175-185, Matsui, et al. (1996) EMBO J.15:2208-2216,Matsui, et al. (1998) J. Cell Biol. 140:647-657,Ishizaki (1997) FEBSLett. 404:18-124), the biological activity that concerns this invention.Therefore, chemical compounds such as Y-27632, any other compound arecovered by this invention as a preferred delivery in a tissue adhesivesystem. Numerous references describing C3 type compounds can be found inMethods in Enzymology, Vol. 256, Part B, Eds.: W. E. Balch, C. H. Der,and A. Hall; Academic Press, 1995, for eg. Pgs. 196-206, 207 et seq,184-189, and 174 et seq. In any event C3 may for example be selectedfrom the group consisting of ADP-ribosyl transferase derived fromClosteridum botulinum and a recombinat ADP-ribosyl transferase.

On the other hand any compound or molecule that does not have a directaction on Rho itself but works to decrease the function of Rho such asanti-sense oligos to Rho, anti-Rho kinase antibodies, and the like. SuchRho antagonists that can be delivered in a tissue adhesive system arealso covered by our invention. The C3 polypeptides of the presentinvention include biologically active fragments and analogs of C3;fragments encompass amino acid sequences having truncations of one ormore amino acids, wherein the truncation may originate from the aminoterminus, carboxy terminus, or from the interior of the protein. Analogsof the invention involve an insertion or a substitution of one or moreamino acids. Fragments and analogs will have the biological property ofC3 that is capable of inactivation Rho GTPases. Also encompassed by theinvention are chimeric polypeptides comprising C3amino acid sequencesfused to heterologous amino acid sequences. Said heterologous sequencesencompass those which, when formed into a chimera with C3 retain one ormore biological or immunological properties of C3. A host celltransformed or transfected with nucleic acids encoding C3 protein or c3chimeric protein are also encompassed by the invention. Any host cellwhich produces a polypeptide having at least one of the biologicalproperties of a C3 may be used. Specific examples include bacterial,yeast, plant, insect or mammalian cells. In addition, C3 protein may beproduced in transgenic animals. Transformed or transfected host cellsand transgenic animals are obtained using materials and methods that areroutinely available to one skilled in the art. Host cells may containnucleic acid sequences having the full-length gene for C3 proteinincluding a leader sequence and a C-terminal membrane anchor sequence(see below) or, alternatively, may contain nucleic acid sequenceslacking one or both of the leader sequence and the C-terminal. membraneanchor sequence. In addition, nucleic acid fragments, variants andanalogs which encode a polypeptide capable of retaining the biologicalactivity of C3 may also be resident in host expression systems.

The Rho antogaonist that is a recombinant proteins can be made accordingto methods present in the art. The proteins of the present invention maybe prepared from bacterial cell extracts, or through the use ofrecombinant techniques. In general, C3 proteins according to theinvention can be produced by transformation (transfection, transduction,or infection) of a host cell with all or part of a C3-encoding DNAfragment in a suitable expression vehicle. Suitable expression vehiclesinclude: plasmids, viral particles, and phage. For insect cells,baculovirus expression vectors are suitable. The entire expressionvehicle, or a part thereof, can be integrated into the host cell genome.In some circumstances, it is desirable to employ an inducible expressionvector.

Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems can be used to provide therecombinant protein. The precise host cell used is not critical to theinvention. The C3 protein can be produced in a prokaryotic host (e.g.,E. coli or B. subtilis) or in a eukaryotic host (e.g., Saccharomyces orPichia; mammalian cells, e.g., COS, NIH 3T3, CHO, BHK, 293, or HeLacells; or insect cells).

Proteins and polypeptides can also be produced by plant cells. For plantcells viral expression, vectors (e.g., cauliflower mosaic virus andtobacco mosaic virus) and plasmid expression vectors (e.g., Ti plasmid)are suitable. Such cells are available from a wide range of sources(e.g., the American Type Culture Collection, Rockland, Md.). The methodsof transformation or transfection and the choice of expression vehiclewill depend on the host system selected.

The host cells harbouring the expression vehicle can be cultured inconventional nutrient media adapted as need for activation of a chosengene, repression of a chosen gene, selection of transformants, oramplification of a chosen gene. One expression system is the mouse 3T3fibroblast host cell transfected with a pMAMneo expression vector(Clontech, Palo Alto, Calif). pMAMneo provides an RSV-LTR enhancerlinked to a dexamethasone-inducible MMTV-LTR promotor, an SV40 origin ofreplication which allows replication in mammalian systems, a selectableneomycin gene, and SV40 splicing and polyadenylation sites. DNA encodinga C3 protein would be inserted into the pMAMneo vector in an orientationdesigned to allow expression. The recombinant C3 protein would beisolated as described below. Other preferable host cells that can beused in conjunction with the pMAMneo expression vehicle include COScells and CHO cells (ATCC Accession Nos. CRL 1650 and CCL 61,respectively).

C3 polypeptides can be produced as fusion proteins. For example,expression vectors can be used to create lacZ fusion proteins. The pGEXvectors can be used to express foreign

polypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can be easily purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.Another stategy to make fusion proteins is to use the His tag system.

In an insect cell expression system, Autographa californica nuclearpolyhedrosis virus AcNPV), which grows in Spodoptera frugiperda cells,is used as a vector to express foreign genes. A C3 coding sequence canbe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter, e.g., the polyhedrin promoter. Successful insertion of a geneencoding a C3 polypeptide or protein will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat encoded by the polyhedrin gene).These recombinant viruses are then used to infect spodoptera frugiperdacells in which the inserted gene is expressed (see, Lehmann et al for anexample of making recombinant MAG protein).

In mammalian host cells, a number of viral-based expression systems canbe utilised. In cases where an adenovirus is used as an expressionvector, the C3 nucleic add sequence can be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene can then be inserted intothe adenovirus genome by in vitro or in vivo recombination. Insertioninto a non-essential region of the viral genome (e.g., region E1 or E3)will result in a recombinant virus that is viable and capable ofexpressing a C3 gene product in infected hosts.

Specific initiation signals may also be required for efficienttranslation of inserted nucleic acid sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire native C3 gene or cDNA, including its own initiation codon andadjacent sequences, is inserted into the appropriate expression vector,no additional translational control signals may be needed. In othercases, exogenous translational control signals, including, perhaps, theATG initiation codon, must be provided. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators.

In addition, a host cell may be chosen which modulates the expression ofthe inserted sequences, or modifies and processes the gene product in aspecific, desired fashion. Such modifications (e.g., glycosylation) andprocessing (e.g., cleavage) of protein products may be important for thefunction of the protein. Different host cells have characteristic andspecific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen to ensure the correct modification andprocessing of the foreign protein expressed. To this end, eukaryotichost cells that possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation of the geneproduct can be used. Such mammalian host cells include, but are notlimited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, W138, and inparticular, choroid plexus cell lines.

Alternatively, a C3 protein can be produced by a stably-transfectedmammalian cell line. A number of vectors suitable for stabletransfection of mammalian cells are available to the public; methods forconstructing such cell lines are also publicly available. In oneexample, cDNA encoding the C3 protein can be cloned into an expressionvector that includes the dihydrofolate reductase (DHFR) gene.Integration of the plasmid and, therefore, the C3 protein-encoding geneinto the host cell chromosome is selected for by including 0.01-300 μMmethotrexate in the cell culture medium (as described in Ausubel et al.,supra). This dominant selection can be accomplished in most cell types.

Recombinant protein expression can be increased by DHFR-mediatedamplification of the transfected gene. Methods for selecting cell linesbearing gene amplifications are known in the art; such methods generallyinvolve extended culture in medium containing gradually increasinglevels of methotrexate. DHFR-containing expression vectors commonly usedfor this purpose include pCVSEII-DHFR and pAdD26SV(A). Any of the hostcells described above or, preferably, a DHFR-deficient CHO cell ligne(e.g., CHO DHFR cells, ATCC Accession No. CRL 9096) are among the hostcells preferred for DHFR selection of a stably-transfected cell line orDHFR-mediated gene amplification.

A number of other selection systems can be used, including but notlimited to the herpes simplex virus thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase, and adeninephosphoribosyltransferase genes can be employed in tk, hgprt, or aprtcells, respectively. In addition, gpt, which confers resistance tomycophendlic acid ; neo, which confers resistance to the aminoglycosideG-418; and hygro, which confers resistance to hygromycin can be used.

Alternatively, any fusion protein can be readily purified by utilisingan antibody specific for the fusion protein being expressed. Forexample, a system described in Janknecht et al. (1981) Proc. Natl. Acad.Sci. USA 88, 8972, allows for the ready purification of non-denaturedfusion proteins expressed in human cell lines. In this system, the geneof interest is subcloned into a vaccinia recombination plasmid such thatthe gene's open reading frame is translationally fused to anamino-terminal tag consisting of six histidine residues. Extracts fromcells infected with recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose columns, and histidine-tagged proteins areselectively eluted with imidazole-containing buffers.

Alternatively, C3 or a portion thereof, can be fused to animmunoglobulin Fc domain. Such a fusion protein can be readily purifiedusing a protein A column.

It is envisioned that small molecule mimetics of the above describedantagonists are also encompassed by the invention.

In the following a method to identify active Rho antagonists will bediscussed.

To test Rho antagonists for activity, a tissue culture bioassay systemwas used. This bioassay is used to define acitivity of Rho antagoniststhat will be effective in promoting axon regeneration in spinal cordinjury, stroke or neurodegenerative disease.

Neurons do not grow neurites on inhibitory myelin substrates. Whenneurons are placed on inhibitory substrates in tissue culture, theyremain rounded. When an effective Rho antagonist is added, the neuronsare able to grow neurites on myelin substrates. The time that it takesfor neurons to growth neurites upon the addition of a Rho antagonist isthe same as if neurons had been plated on growth permissive substratesuch as laminin or polylysine, typically 1 to 2 days in cell culture.The results can be scored visually. If needed, a quantitative assessmentof neurite growth can be performed. This involved measuring the neuritelength in a) control cultures where neurons are plated on myelinsubstrates and left untreated b) in positive control cultures, such asneurons plated on polylysine c) or treating cultures with differentconcentrations of the test antagonist.

To test C3 in tissue culture, it has been found that the bestconcentration is 25-50 ug/ml. Thus, high concentrations of this Rhoantagonist are needed as compared to the growth factors used tostimulate neurite outgrowth. Growth factors, such as nerve growth factor(NGF) are used at concentrations of 1-100 ng/ml in tissue culture.However, growth factors are not able to overcome growth inhibition bymyelin. Our tissue culture experiments are all performed in the presenceof the growth factor BDNF for retinal ganglion cells, or NGF for PC12cells. When growth factors have been tested in vivo, typically thehighest concentrations possible are used, in the ug/ml range. Also theyare often added to the CNS with the use of pumps for prolonged delivery(eg. Ramer et al, IBID). For in vivo experiments the highestconcentrations possible was used when working with C3 stored as a frozen1 mg/ml solution. The concentration that was chosen does not prevent thefibrin matrix from polymerizing.

For test purposes it was decided to dilute a 1 mg/ml solution of C3 with1/3 volume thrombin and 1/3 volume fibrinogen solutions (contain calciumand aprotinin). In order to increase the concentration of C3, it wouldbe possible to lyophylize C3 and then resuspend it in the fibrinogensolution. Lyophilized C3 has been tested and found to be active.

The Rho antagonist C3 is stable at 37 C for at least 24 hours. Thestability of C3 was tested in tissue culture with the followingexperiment. The C3 was diluted in tissue culture medium, left in theincubator at 37C for 24 hours, then added to the bioassay systemdescribed above, using retinal ganglion cells as the test cell type.These cells were able to extend neurites on inhibitory substrates whentreated with C3 stored for 24 hours at 37C. Therefore, the minimunstability is 24 hours. This is in keeping with the stability projection,based on amino acid composition (see sequence data, below).

In the following various tissue Adhesives and Formulations used to makethem will be discussed.

Different types of tissue adhesive can be made. Examples includecollagen gels, fibrin tissue adhesives. Other examples are matrigel,laminin networks, and adhesives based on a composition of basmentmembrane proteins that contain collagen.

Fibrin sealant has three basic components: fibrinogen concentrate,calcium chloride and thrombin. Other components can be added to affectthe time of clot formation, and the size of the protein network that isformed. Generally when the components mix, a fibrin coagulum is formedin that the fibrinogen molecule is cleaved through the action ofthrombin to form fibrin monomers which spontaneously will polymerize toform a three-dimensional network of fibrin, largely kept together byhydrogen bonding. This corresponds to the last phase of the naturalblood clotting cascade, the coagulation rate being dependent on theconcentration of thrombin used. In order to improve the tensilestrength, covalent crosslinking between the fibrin chains is providedfor by including Factor XIII in the sealant composition. In the presenceof calcium ions, thrombin activates factor XIII to factor XIIIa.Activated factor XIIIa together with thrombin catalyzes thecross-linkage of fibrin and increases the strength of the clot. Thestrength of the fibrin clot is further improved by the addition offibronectin to the composition, the fibronectin being crosslinked andbound to the fibrin network formed. During wound healing the clotmaterial undergoes gradual lysis and is completely absorbed.

To prevent a too early degradation of the fibrin clot by fibrinolys, thefibrin sealant composition may comprise a plasminogen activatorinhibitor or a plasmin inhibitor, such as aprotinin. Such an inhibitorwill also reduce the fibrinolytic activity resulting from any residualplasminogen in the fibrinogen composition. Similarly, compositions mayinclude hyaluronic acid (or other polysaccharides), and these may alsocomprise a hyaluronidase inhibitor such as one or more flavonoids (orcorresponding inhibitors for other polysaccharides) in order to preventdegradation (i.e. to prolong the duration) of the hyaluronic acidcomponent by hyaluronidase which is always present in the surroundingtissues. The hyaluronic acid may, as mentioned above, be crosslinked, acommercially available example being Hylan.RTM. (trademark, availablefrom Biomatrix, Ritchfield, N.Y., USA). The hyaluronic acid compositionsmay e.g. have the form of gels, solutions, etc.

Fibrin clots in any one of the above described embodiments, may be usedfor the application of a pharmaceutically active substance. Byincorporating a drug, such as an antibiotic, a growth factor, etc. intothe tissue adhesive it will be enclosed in the fibrin network formedupon application of the tissue adhesive. It will thereby be ensured thatthe drug is kept at the site of application while being controllablyreleased from the composition.

Fibrin sealant products prepared from human plasma fibrinogen/FactorXIII are available commercially. One product is a tissue glue calledTisseel Fibrin Sealant (Baxter Hyland Immuno Corporation).(Tissucol/Tisseel, Immuno AG, Vienna) and another Beriplast P, Hoechst,West Germany. A frozen formution of a fibrin glue delivered with a 2syringe system is Hemaseel made by Hemacure Inc. (Kirkland, Quebec).

In the following methods for making Tissue Adhesive Delivery kits willbe discussed.

In a preferred embodiment, the kit includes the solutions provided inseparate bottles to prevent mixing before use, and an applicatordesigned so as to permit mixing of the fibrinogen/Factor XIII andthrombin with C3 at the body site. The kit would provide pre-measuredamounts of the fibrinogen and factor XIII in one bottle, the thrombin inanother bottle, a calcium chloride solution in third bottle, and a C3solution in a fourth bottle. The contents of the bottles would be mixedin a prescribed order, as detailed in the example below. The kit canalso include one or more other storage containers which are anynecessary reagents including solvents, buffers, etc. The kit could besold as lyophilized or frozen components to preserve the activity of C3or other Rho antagonist added to the kit.

The applicator can, for example, take the form of a glass or plasticsyringe with disposable needles. With a single syringe system, thecomponents of the kit would be mixed immediately before application tothe injury site.

A more elaborate system would allow two syringes to be attached, so thatthe mixing could, take place in the syringe°or a mixing compartment ofthe syringe, before injection. One example of a two syringe system is aLuer lock syringe, such as used for mixing adjuvants. For this a 3-waystopcocks, such as commercially available (Bio-Rad cat #7328103) isattached to the syringe so that the solution can be passed back andforth beore attaching the injection needle to the third port of the3-way stopcock. These are plastic, sterile, and disposable.

Another method of application could be through the use of a clip to holdtwo syringes, and the clip would have a common plunger to ensure thatequal volumes of the thrombin and fibrinogen components are mixed in achamber with the calcium chloride and C3, before being ejected troughthe needle.

Other Ingredients for the Tissue Adhesive Rho Antagonist Delivery Systemare discussed hereinafter,

Other components can be added to the tissue adhesive to improve efficacyof the treatments. Such additions include growth factors, proteaseinhibitors, cytokines, anti-inflammatory compounds, cell transplantsystems. Agents that prevent cell death, such as agents that affect theapoptosis pathway could be added components to the delivery system.

Methods of Packaging Delivery System are discussed hereinafter.

In the preferred formulation, Rho antagonist, fibrinogen and thrombinare mixed together just before application, so that polymerization ofthe gel occurs in the injured CNS. Therefore, it is important that thefibrinogen and thrombin are package separately. However, the C3 can bepackaged separately, or added to either the thrombin or fibrinogenbottles. In another formulation, the fibrinogen, thrombin and C3 arepackaged together, but help at low pH, which prevents polymerization ofthe gel. Polymerization would be induced by mixing this formution with abasic component that would neutralize the pH to induce coagulation ofthe adhesive. In another formultion, the Rho antogonist could be addedseparately to the fibrinogen/thrombin mix in the form of liposomes orother similar delivery system. Living cells could that secrete C3 couldbe added as Rho antagonist.

A method of Applying Rho antagonist in vivo is discussed hereinafter.

Tissue adhesive formulations are typically applied to wound sites with asyringe and needle. The shape of the need determine the type of surfacethat is formed when the adhesive polymerizes. In some cases, adhesivescan be sprayed onto the wound surface, or into the desired region. Thisinvention covers all types of syringes and needles used to apply fibrinplus Rho antagonists to injured regions of the CNS. In addition, itcovers the addition of previously polymerized tissue adhesives with C3to the wound. For example, fibrin can be polymerized in a teat tube, andforcepts used to remove the gel and place it in the body cavity.Similarly, collagen can be applied by pre-polymerization and applicationby using focepts to place the gel in the injured spinal cord. Oneexample of this is more fully explained in the example section of thisapplication.

Tests were done with Gelfoam(TM), a surgical collagen-based sponge, andElvax, a slow release plastic (Lehmann et al 1999, IBID) for the abilityto deliver biologically effective concentrations of C3. Neither of thesetwo delivery systems was effective. Therefore, only tissue adhesiveformulations (i.e. the matrix forming formulations discussed herein)have efficacy in the delivery of C3 to the injured CNS in vivo.

Therapeutic Applications/Medical Uses will be discussed below.

The tissue adhesive system for the delivery of Rho antoagonists may beuseful in many other conditions that affect the central and peripheralnervous system. Treatments that are effective in eliciting sproutingfrom injured axons are equally effective in treating some types ofstroke (Boston life sciences, Sep. 6, 2000 Press Release). Since it hasbeen determined that it is possible to elicit sprouting (using akit ofthe present invention), it is obvious that the treatments can beextended to stroke. Similarly, although the subject of this invention isrelated to delivery of Rho antagonists to the traumatically damagednervous system, this invention also pertains to damage fromneurodegeneration, such as during Parkinson's disease, Alzheimer'sdisease, prion diseases or other diseases of the CNS were axons aredamaged in the CNS environment. In such cases, small volumes of thetissue adhesive with r C3 could be injected into the affected regionwith the use of a syringe. The treatment will cause local sprouting torestore function of neurons whose axon processes had retracted in thecourse of the neurodegeneration.

Testing example Formulation(s) and Delivery System(s) will be DiscussedBelow.

Tests of invention t formulation were conducted in mice after injury ofthe corticospinal tract. All mice were tested for anatomicalregeneration of lesioned axons by anterograde tracing techniques. Someof the mice were also assessed for recovery of locomotion. The detailsof these experiments are given in the experimental section, the examplesections, and the results are shown in the figures.

EXAMPLES Example 1 A Kit for a Tissue Adhesive System

The kit contains:

-   1 vial fibringen-   1 vial apropinin solution for reconsitution of fibrinogen-   1 vial thrombin-   1 vial calcium chloride solution for reconsitution of thrombin-   1 vial C3 solution

1.1 Lyophilized fibrinogen (75 mg/ml) in glycine buffer (2 mg/ml NaO, 4mg/ml trisodium citrate, 15 mg/ml glycine) was reconstituted in anaprotinin solution 3000 KIU/ml and heated to 37 C. For ease of handling,a combined heating and stirring device was used (appropriate vialscontain a maganetic stirrer. This is called solution I.

1.2 A thrombin solution is prepared; the solution comprisin Lyophilizedthrombin 500 IU/ml , 2.4 mg/ml glycine, 8 mg/ml sodium chloride. Thecalcium chloride solution (40 umol CaCl2) and thrombin are mixed andheated to 37C. This is called solution II.

1.3 A solution of C3 (1 mg/ml) is heated to 37C

1.4 Equal amounts of solution I, II, and III are mixed, and immediatelydrawn up in a syringe, and added to the injury site where polymerizationoccurs. Thus the C3 is added as part of the fibrin glue solution that isplaced in the lesion cavity to polymerize.

A combined heating and stirring device can be used in conjunction withthe kit. For this, small magnetic stirrers are included in each of themixing vials. The vials are then placed in the combined mixing andwarming device where the magnetic stirrer keeps the solution stirredwhile the solution is warming.

Mice that received a dorsal hemisection were treated with the fibrin/C3adhesive. In some experiments, 10 μl of 1 mg/ml C3 in phosphate bufferedsaline was added to the lesion site before applying the C3/fibrin.Behavior recovery was assessed in an open field environment as describedby Beattie, Basso and Breshnahan (1995) J. Neurotrauma 12:1-20.Anatomical regeneration was assessed by anterograde labeling of thecorticospinal fibres.Three weeks to three months after injury, thecorticospinal fibres were labeled by inject the anterograde tracerWGA-HRP into the motor cortex as described in the art (Huang(1999)Submitted.). Two days later the animals were killed, the spinalcord removed, and longitudinal sections cut and reacted for HRPenzymatic activity, as described (Huang (1999)Submitted.). The labeledfibres were observed by microscopy to extend many mm past the lesionsite (see FIGS. 5 and 6) after treatment with C3/fibrin.

Example 2 Modification of the Kit in Example 1

The formulation given in example 1 was used with the followingmodifications. Solution II is made with the addition of recombinant C3directly to the solution II vial. In other words, solution II containsthrombin, calcium chloride and C3. Solution I is loaded in one syringe,solution II is loaded in a second syringe. A syringe with a plunger thatsimultaneously loads both solutions is used. Thus the solutions aremixed as they enter a small chamber before the needle, and thepolymerization occurs in situ in the injured region of the CNS where thesolution is applied. The system describe here is the Duploject systemfrom Baxter Pharmaceuticals U.S.A.

Example 3 Modification of the Kit in Example 1

As example 2, but the C3 solution is mixed in vial I with thefibrinogen. Vial one and vial II are heated and prepared as described inexample 1, and injected into the injured CNS with the Duploject system.

Example 4 Collagen Gels Used a a Tissue Adhesives

First collagen is purified. Collagen can be purified from any source,human or mammalian. One source of collagen is the EHS tumor cell linewhich is passed in mice. Collagen was purified from rat tails. The tailswere soaked in 70% alcohol for about 20 minutes. The remaining stepswere performed under aseptic conditions. The tails are broken about 2 cmfrom the tip with a hemostat and the tendon is slowly pulled out andplaced in a sterile dish. The tendons are cut into small pieces andsoaked in acetic acid-water (1:1000) for 48 hours in the cold. 150 ml ofsolution is used per tail. The solution is centrifuged at 15,0000 rpm,30 min. and stored in aliquots at B10C.

Collagen gels with C3 as Rho antagonist are formed in vivo as follows.For treatment of one mouse, 40 μg of C3 was lyophilized. The C3 proteinwas reconstituted in 10 u 1 of 7.5% NaHCO₃. Collagen at 0.7 mg/ml wasused, and 25 μl collagen was added to the C3 solution. A mouse that hadreceived a dorsal hemisection of the spinal cord was treated with 10 μlof 1 mg/ml C3 in the collagen (i.e. at the lesion site). The time ittakes for the collagen to polymerize may be modified by varying theNaHCO₃ solution. Anatomical regeneration of transected cortical spinalfibres was assessed as described in the detailed description of theinvention.

Example 5

Procedure to Make recombinant C3 as a Rho antoagonist. RecombinantC3protein was made as follows. The plasmid pGEX2T-C3 coding for theglutathione-S-transferase (GST)-C3 fusion protein was obtained from N.Lamarche (McGill Univ.). Bacteria were transformed with pGEX2T-C3,allowed to grow overnight induced with IPTG, and sonicated to break openthe cells. The recombinant protein was purified by affinitychromotography as described (Ridley and Hall (1992) Cell. 70:389-399).The GST fusion protein was cleaved by thrombin, and thrombin was removedby incubation with 100 μl of p-aminobenzamidine agarose-beads (Sigma).The C3 solution was dialyzed against PBS, and sterilized with a 0.22 μmfilter. The C3 concentration was evaluated by protein assay (DC assay,BioRad Labs, Missassauga, Ont.) and C3 purity was controlled by SDS-PAGEanalysis.

Example 6 Testing the Fibrin-Rho-Antagonist Formulation Using theDelivery System

To test the tissue adhesive system a rodent model of spinal cord injurywas used. For this, Balb-c mice were anaesthetized with 0.6 ml/kghypnorm, 2.5 mg/kg diazepam and 35 mg/kg ketamine. A section of thethoracic spinal cord was exposed using fine rongers to remove the bone.A dorsal hemisection was made to cut the dorsal columns at level T6. Thefibrin/C3 adhesive was injected immediately after injury. As controlanother group of animals received fibrin alone, and a third groupreceived no treatment. The following day behavioural testing began, andcontinued for three weeks. The animals were placed in an open fieldenvironment that consisted of a rubber mat approximately 4′×3′ in size.The animals were left to move randomly, the movement of the animals werevideotaped. For each test two observers scored the animals for abilityto move ankle, knee and hip joints in the early phase of recovery. Inthe intermediate, phase, the ability to support weight and correctplacement of the feet was assessed (dorsal or plantar placement). In thelate phase of recovery, the animals were assessed for correct footposition, trunk stability, and foot drag. Only animals that receivedC3/fibrin reached the late phase of recovery of coordinatedforelimb-hindlimb movement. Untreated control animals did not typicallypass beyond the early phase of recovery.

Additional Experimental Activity will be Discussed Below.

Spinal Cord Injury

To study The CST was cut bilaterally by a dorsal hemisection extendingpast the central canal (1 (FIG. 2) at the T6 level. Balb-c mice wereanaesthetized with 0.6 ml/kg hypnorm, 2.5 mg/kg diazepam and 35 mg/kgketamine. A section of the thoracic spinal cord was exposed using finerongers to remove the bone, and a dorsal hemisection was made at levelT6. Fine sissors were used to cut the dorsal half of the spinal cord,and it was recut a second time with fine knife to ensure all lesionsextended past the central canal. Three weeks to four weeks after injury,the corticospinal fibres were labeled by injection the anterogradetracer WGA-HRP into the motor cortex as into 6 sites. For injection intothe motor cortex a pulled glass pipette was used. Two days later theanimals were perfused transcardially with saline then 4%paraformaldehyde and the spinal cords and brains were removed.

C3 toxin was delivered locally to the site of the lesion by afibrin-based tissue adhesive delivery system (FIG. 1). Recombinant C3was mixed with fibrinogen and thrombin in the presence of CaCl₂.Fibrinogen is cleaved by thrombin, and the resulting fibrin monomerspolymerize into a three-dimensional matrix. C3 was added as part of afibrin adhesive, which polymerized within about 10 seconds after beingplaced in the injured spinal cord. Anterograde tracing with WGA-HRP wasused to study anatomical regeneration past the site of lesion in threegroups of animals: animals treated with fibrin plus C3 (C3/fibrin),animals treated with fibrin alone, and animals that did not receivedtreatment after injury (see FIG. 7). With no treatment, transected CSTaxons retract back from the site of lesion from 500 um to 1 mm (FIG. 3).Animals treated with fibrin alone showed less axon retraction, andsprouting of axons was observed to extend towards the scar. Applicationof C3 to the injured spinal cord elicited an extensive sprouting of CSTaxons into the dorsal white matter, and the axons grew into the scar andand extended past past the lesion (FIG. 4). A long distance regenerationof individual CST axons and axon bundles was elicited by C3 (FIG. 5),but not in untreated or fibrin controls. This regeneration wassignificantly different from any growth observed following treatmentwith fibrin alone.

Several different tissue adhesive delivery systems were tested. When C3was delivered in collagen gels less axon retraction was observed, butthe same extent of axon regeneration was not observed as with fibrin.Gelfoam(TM), a surgical collagen, sponge, was also tested. Gelfoam wasnot as effective as fibrin as promoting long-distance regeneration (FIG.7). A non-biological material, Elvax, was also tested which is apolymer-based artificial release system (see Lehmann et al, 1999 IBID).This system was not effective in allowing cut axons access to C3.

To test functional recovery following treatment of injured spinal cordwith C3, three groups of animals were score for locomotor behaviour inan open field environment according to the 21 point BBB scale (Basso etal. ). The animals were examined by two reviewers and were placed alonein an open field environment that consisted of a rubber matapproximately 4′×3′ in size. Each animal was videotaped for approx. 3min. For the early and intermediate phases, the BBB scores were derivedfollowing observation, and confirmed by video analysis. In the latephase of recovery, the BBB score was determined from the . videosprojected on a computer at 3 speed from sequences of 4 steps or more.The BBB test includes three phases of recovery: an early phase (scores1-7) of joint movement, an intermediate phase (score 8-13) where weightsupport and foot placement (dorsal or plantar) are assessed, and a latephase of coordinated movements (scores 14-21) where correct footposition, and foot drag are examined. The C3 treated animals rapidlyregained the ability to support weight (FIG. 7B) while control animalsmoved mostly by the action of their forelimbs (FIG. 7A). The controlgroups entered the intermediate recovery phase with the ability tosupport weight within one weeks, at which point they obtained theirrecovery plateau. Animals that received C3 treatment continues torecover over the 1 month period of observation, and recoveredcoordinated movement and almost normal stepping (FIG. 8).

In rats that receive a contusion injury the recovery period depends onthe severity and location of the lesion. Typically, rats reach a plateauof recovery by about two week, whereas after dorsal hemisection in miceit was found that the plateau of recovery is reached within about 1week. The remarkable improvement in C3-treated mice within one day ofspinal cord lesion is likely due to changes in the local spinal cordcircuitry. These local changes might result from the robust sproutingimmediately after application of C3 is applied to the transected axons.Rates of axon growth in vivo are known to be approximately the same asthe slow axonal transport rate of 50-200 um/hr. It is also possible thatthe local effects on the spinal cord are mechanistically different byacting on central pattern generators implicated in walking behaviors orby neuroprotection immediatley after treatment. Most importantly,treated mice performed better immediately after lesion, and theyrecovered almost normal walking patterns by one month (FIG. 8). Thisslower phase of recovery is attributed to the long-distance regenerationof axons that was induced by C3 (FIG. 4). Moreover, while we only flowedthe CST axons in this study, our treatments also are likely stimulategrowth from other transected axonal populations.

In the Following Production of Recombinant C3 will be Discussed.

C3 is a protein product made by the bacteria Clostridium botulinum. Thefragment containing the C3 gene was cloned into a pGEX vector (fromAmersham Pharmacia Biotech inc. Baie D'rfe, Quebec, Canada), nowreferred to as pGEX2T-C3, and this vetor was obtained from NathalieLamarche of McGill University. To confirm the C3 sequence correspondedto that reported in the literature the insert was sequenced (seesequence below). The C3-containing pGEX vector was transformed into theRR1 strain of E. coli (GIBCO).

Bacteria were grown in L-Broth (10 g/L Bacto-Tryptone, 5 g/L YeastExtract, 10 g/L NaCl (Fisher Scientific) with Ampicillin(BMC-Roche) at50 ug/ml in a shaking incubator for 1 hr at 37° C. Isopropylβ-D-thiogalactopyranoside(IPTG), (GIBCO) was added to a finalconcentration of 0.5 mM to induce production of recombinant protein andthe culture was grown for a further 6 hrs at 37 C. Bacterial pelletswere obtained by centrifugation, in 250 ml centrifuge bottles, at 6000rpm at 4° C. for 5 min. Pellets can be kept frozen at −80° C. at thistime.

5 mls of Buffer A(50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mMDTT)+1 mM PMSF was added to each pellet. Pellets were resuspended andtransferred to a 50 ml plastic beaker on ice, and a further 5 mls ofbuffer A was used to wash the centrifuge bottles. Total volume of bufferA+pellets from a 2 L culture is usually 30-40 mls. The pellets, on ice,were sonicated 5×30 sees using a BRANSON SONIFIER 450 probe sonicator.Bacteria were cooled on ice 1 minute between sonications. The sonicatewas centrifuged in a Sorvall SS-34 rotor at 10,000 rpm for 10 min at 4 Cto clarify supernatant.

Glutathione-agarose beads (SIGMA#G-4510) were purchased as a lyophilizedpowder and the beads were swollen in deionized water, then stored in 1 MNaCl at 4° C. Five ml of the beads (50% v/v) were washed in a 50 ml tubefilled with buffer A (no PMSF). Tube was centrifuged at 2000 rpm (500 g)for 5 min, water was removed, and replaced with buffer A. These beadswere added to the cleared bacterial supernatant, and mixed for 1-2 hrsat 4 C. The beads were washed 4 times with buffer B (buffer A, NaCl is150 mM, no PMSF), then 2× with buffer C (buffer B+2.5 mM CaCl2). Washeswere poured out and the beads retained each time. Next, 5 mls ofthrombin(B6vine, Plasrainogen-free, CALBIOCHEM #605160) 20 U at (50% v/v) was added to the beads to cleave the C3 from the GST affinitypurification tag (see cleavage site in the nucleotide sequnce givenbelow). This reaction was left overnight, with mixing, at 4° C.

The beads are loaded into an empty 10 ml column, PBS (phosphate bufferedsaline) was added to the column and 20 1 ml aliquots were collected. Todetermine the location of the protein peak, 0.5 μl spots were put on anitrocellulose sheet, from each aliquot, and this is stained with AmidoBlack(Bio-rad) as a protein dot-blot. Aliquots containing C3 were pooledand 20 uls p-Aminobenzamidine (SIGMA#A7155) was added. The solution wasmixed for 30min at 4 C to remove the thrombin. The C3 is centrifuged toremove the p-aminobenzamidine, and then concentrated using aCENTRIPREP-10 concentrator (AMICON). The concentrated C3 is then passedthrough a PD-10 column (PHARMACIA, containing Sephadex G-2SM) and 10 0.5ml aliquots are collected. A dot-blot is done on these aliquots, and theappropriate aliquots (usually 3 tubes) are pooled (total volume about1.5 mls). The purified recombinant protein was filter-sterilized,aliquoted, and stored at 80° C. A protein assay was done on a smallamount to determine precisely the concentration. Purity of therecombinant C3 was evaluated by SDS polyacrylamide gel electrophoresis.Bioactivity was assed with a bioassay using either retinal ganglioncells or PC12 cells (see identification of Rho antagonist section).

SEQUENCE of (Known) Rho Antagonist C3 Used in the Experiments

Nucleotide sequence including part of the plasmid GST sequence. Thevector with the GST sequence is commercially available and thus theentire GST sequence including the start was not sequenced. It wasdesired to determine only the sequence 3′ to the thrombin cleavage sitewhich releases C3 from the GST sequence. The thrombine cleavage site isshown with an arrow and is located just to the left of the underlinednucleotide sequence below (i.e. the arrow shows the thrombin cleavagesite). The underlined sequence shows additional coding sequencetranslated in our recombinant protein that is not reported in theliterature.

Both strands were sequenced to verify that there were no errors in thesequence.

5′ GTG GCG ACC CTT CCC AAA TCG GAT CTG GTT CCG CGTGGA TCC TCT AGA GTC GAC CTG CAG GCA TGC AAT GCT TAT TCC ATT AAT CAA AAGGCT TAT TCA AAT ACT TAC CAG GAG TTT ACT AAT ATT GAT CAA GCA AAA GCT TGGGGT AAT GCT CAG TAT AAA AAG TAT GGA CTA AGC AAA TCA GAA AAA GAA GCT ATAGTA TCA TAT ACT AAA AGC GCT AGT GAA ATA AAT GGA AAG CTA AGA CAA AAT AAGGGA GTT ATC AAT GGA TTT CCT TCA AAT TTA ATA AAA CAA GTT GAA CTT TTA GATAAA TCT TTT AAT AAA ATG AAG ACC CCT GAA AAT ATT ATG TTA TTT AGA GGC GACGAC CCT GCT TAT TTA GGA ACA GAA TTT CAA AAC ACT CTT CTT AAT TCA AAT GGTACA ATT AAT AAA ACG GCT TTT GAA AAG GCT AAA GCT AAG TTT TTA AAT AAA GATAGA CTT GAA TAT GGA TAT ATT AGT ACT TCA TTA ATG AAT GTT TCT CAA TTT GCAGGA AGA CCA ATT ATT ACA AAA TTT AAA GTA GCA AAA GGC TCA AAG GCA GGA TATATT GAC CCT ATT AGT GCT TTT CAG GGA CAA CTT GAA ATG TTG CTT CCT AGA CATAGT ACT TAT CAT ATA GAC GAT ATG AGA TTG TCT TCT GAT GGT AAA CAA ATA ATAATT ACA GCA ACA ATG ATG GGC ACA GCT ATC AAT CCT AAA TAA 3′

Nucleotide sequence of recombinant C3 protein: the sequence given belowrepresents the entire coding sequence for the Rho antagonist used in theexperments mentioned herein. It is similar to the sequence shown abovebut does not include the GST portion which when the protein is made isenzymatically removed with thrombin.

  1 GGATCCTCTA GAGTCGACCT GCAGGCATGC AATGCTTATT CCATTAATCA  51AAAGGCTTAT TCAAATACTT ACCAGGAGTT TACTAATATT GATCAAGCAA 101 AAGCTTGGGGTAATGCTCAG TATAAAAAGT ATGGACTAAG CAAATCAGAA 151 AAAGAAGCTA TAGTATCATATACTAAAAGC GCTAGTGAAA TAAATGGAAA 201 GCTAAGACAA AATAAGGGAG TTATCAATGGATTTCCTTCA AATTTAATAA 251 AACAAGTTGA ACTTTTAGAT AAATCTTTTA ATAAAATGAAGACCCCTGAA 301 AATATTATGT TATTTAGAGG CGACGACCCT GCTTATTTAG GAACAGAATT351 TCAAAACACT CTTCTTAATT CAAATGGTAC AATTAATAAA ACGGCTTTTG 401AAAAGGCTAA AGCTAAGTTT TTAAATAAAG ATAGACTTGA ATATGGATAT 451 ATTAGTACTTCATTAATGAA TGTTTCTCAA TTTGCAGGAA GACCAATTAT 501 TACAAAATTT AAAGTAGCAAAAGGCTCAAA GGCAGGATAT ATTGACCCTA 551 TTAGTGCTTT TCAGGGACAA CTTGAAATGTTGCTTCCTAG ACATAGTACT 601 TATCATATAG ACGATATGAG ATTGTCTTCT GATGGTAAACAAATAATAAT 651 TACAGCAACA ATGATGGGCA CAGCTATCAA TCCTAAATAA

Amino Acid Sequence (One Letter Code)

Translation of the above sequence to show amino acid sequence. Aminoacids in bold, highlight differences from published sequence (Popoff etal. (1990) Nucl. Acid. Ress. 18:1291. EMBL accession no. X511464.) The11 N-terminal sequences are additional; there is a single amino acidchange of an alanine (hydrophobic) to glutamic acid (Q).

GSSRVDLQAC NAYSINQKAY SNTYQEFTNI DQAKAWGNAQ YKKYGLSKSE KEAIVSYTKSASEINGKLRQ NKGVINGFPS NLIKQVELLD KSFNKMKTPE NIMLFXGDDP AYLGTEFQNTLLNSNGTINK TAFEKAKAKF LNXDRLEYGY ISTSLMNVSQ FAGRPIITKF KVAKGSKAGYIDPISAFQGQ LEMLLPRHST YHIDDMRLSS DGKQIIITAT MMGTAINPK

Number of amino acids: 229

Molecular weight: 25507.5

Theoretical pI: 9.43

Amino acid composition:

Ala (A) 18 7.9% Arg (R) 6 2.6% Asn (N) 18 7.9% Asp (D) 10 4.4% Cys (C) 10.4% Gln (Q) 12 5.2% Glu (E) 10 4.4% Gly (G) 16 7.0% His (H) 2 0.9% Ile(I) 18 7.9% Leu (L) 17 7.4% Lys (K) 23 10.0% Met (M) 7 3.1% Phe (F) 104.4% Pro (P) 7 3.1% Ser (S) 20 8.7% Thr (T) 14 6.1% Trp (W) 1 0.4% Tyr(Y) 11 4.8% Val (V) 6 2.6% Asx (B) 0 0.0% Glx (Z) 0 0.0% Xaa (X) 2 0.9%

Total number of negatively charged residues (Asp+Glu): 20

Total number of positively charged residues (Arg+Lys): 29

Estimated half-life:

The N-terminal of the sequence considered is G (Gly).

The estimated half-life is: 30 hours (mammalian reticulocytes, invitro).

-   -   >20 hours (yeast, in vivo).    -   >10 hours (Escherichia coli, in vivo).

Instability index:

The instability index (II) is computed to be 26.88

This classifies the protein as stable.

Aliphatic index: 75.07

Grand average of hydropathicity (GRAVY): −0.479

Turning now to FIG. 9, this figure illustrates in schematic fashion asystem exploiting a kit of the present invention for mixing anddelivering a supplemented matrix forming material. An actual apparatusmay for example be of multi-cartridge syringe type as known or modifiedas necessary or desired.

The kit portion of the illustrated system comprises a container mean 1for fibrinogen material, a container means 2 for thrombin material and acontainer means 4 for a therapeutically active agent for facilitatingaxon growth (e.g. C3 or a modified or hybrid C3). If desired ornecessary the the kit portion may include additional containers for theseparate containment of other desired or necessary components; as shownthe system in FIG. includes in dotted outline an additional containermeans for the flowable matrix forming part of the kit. The system alsoincludes a mixing container 6 wherein the C3 (hybrid) is mixed with thematrix farming elements to form the supplemented flowable matrix formingcarrier. The feed line 8 is indicative of the addition of C3 to thecontainer 8 whereas the feed line 10 is indicative of the addition ofthe flowable matrix forming elements from containers 1 and 2 and whichis formed from the merging of feed lines 12 and 13. The mixing in thecontainer means 6 may be effected or carried out in any suitable (known)fashion, (e.g. simple stirring with a magnetic stirrer. The output line15 of the mixing container is indicative of the delivery of thesupplemented mixture to the lesion site (e.g. by needle (e.g. syringe),pipette, etc.

Although in FIG. 9 the therapeutically active agent for facilitatingaxon growth (e.g. C3) is shown As being associated with a separatecontainer 4, if so desired or as necessary the therapeutically activeagent may be associated with a container holding a flowable carriercomponent (e.g. a container may hold fibrinogen and C3).

1. An axon growth stimulation kit comprising a first container means forcontaining a flowable carrier component or two or more separatecomponents capable once intermingled of forming a flowable carriercomponent, said flowable carrier components each being capable offorming a therapeutically acceptable matrix in vivo at a nerve lesionsite and a second container means for containing a therapeuticallyactive agent for facilitating axon r growth at the lesion site whereinsaid therapeutically active agent is releasable from said in vivo matrixinto the adjacent external environment.
 2. An axon growth stimulationkit as defined in claim 1 comprising means for dispersing thetherapeutically active agent in said flowable carrier component so as toform a flowable axon growth stimulation composition and means fordeliverying the flowable axon growth stimulation composition to thelesion site.
 3. An axon growth stimulation kit as defined in claim 1wherein said therapeutically . acceptable matrix is a collagen matrix.4. An axon growth stimulation kit as defined in claim 1 wherein saidtherapeutically acceptable matrix is a fibrin matrix.
 5. A biocompatiblecomposition comprising: (i) at least one supplement selected from thegroup consisting of therapeutically active agents for facilitating axongrowth; and (ii) a flowable carrier component capable of forming atherapeuticallly acceptable matrix in vivo at a nerve lesion site;wherein said supplement is releasable from said matrix into the adjacentexternal environment.
 6. A biocompatible composition as defined in claim5 wherein said therapeutically acceptable matrix is a collagen matrix. ;7. A biocompatible composition as defined in claim 5 wherein saidtherapeutically acceptable matrix is a fibrin matrix.
 8. A method forthe preparation of a flowable biocompatible composition comprisingadmixing (i) at least one supplement selected from the group consistingof therapeutically active agents for facilitating axon growth and (ii) aflowable carrier component capable of forming a therapeuticalllyacceptable matrix in vivo at a nerve lesion site; wherein saidsupplement is releasable from said matrix into the adjacent externalenvironment.
 9. A method as defined in claim 8 wherein saidtherapeutically acceptable matrix is a. collagen matrix.
 10. A method asdefined in claim 8 wherein said, therapeutically acceptable matrix is afibrin matrix.